右美托咪定对坏死性小肠结肠炎新生大鼠丙泊酚麻醉后海马神经元的影响

目的探讨右美托咪定对坏死性小肠结肠炎新生大鼠丙泊酚麻醉后海马神经元的影响。方法取30只出生1日的新生SD大鼠,制作成坏死性小肠结肠炎模型。断头取脑,分离两侧海马组织,将原代培养6 d的海马神经元随机分为空白对照组(C组)、丙泊酚组(P组)、丙泊酚加右美托咪定组(PD组),每组10只。P组加入丙泊酚,使终浓度为100μmol/L; PD组加入右美托咪定和丙泊酚,使终浓度分别为100 nmol/L和100μrnol/L; C组加入等体积生理盐水。培养6 d,显微镜下观察神经元生长情况,比较3组神经元平均突起总长度。用免疫荧光化学技术标记神经元和星形胶质细胞,比较3组平均荧光密度。细胞经各种处理后,收集并裂解,提取细胞总蛋白,检测蛋白含量(BCA法)。取50μg待测蛋白质,Western blot法测定神经元pAkt和Bcl-2蛋白水平。结果 C组的神经元平均突起总长度与突触素平均荧光密度分别为(133. 20±34. 01)μm与(30. 57±7.85),为3组中最高,显著高于P组的(83.21±18.96)μm与(14.67±4.25)(P0.05); PD组的神经元平均突起总长度与突触素平均荧光密度分别为(107.11±23.02)μm与(24.42±6.94),与C组比较无统计学意义(P0.05),但显著高于P组(P0.05)。C组的Bal-2蛋白与p Akt蛋白水平分别为(0.72±0.21)与(0.85±0.22),均为3组中最高,显著高于P组的(0.19±0.05)与(0.41±0.11)(P0.05); PD组Bal-2蛋白、pAkt蛋白水平分别为(0.56±0.18)与(0.78±0.19),与C组比较无统计学意义(P 0.05),但显著高于P组(P0.05)。结论丙泊酚会影响坏死性小肠结肠炎新生大鼠的海马神经元突起及突触发育,右美托咪定具有一定的神经元保护作用,可抑制丙泊酚对海马神经元突起及突触发育的影响,并且右美托咪定对神经元的保护作用可能与激活P13K-Akt-Bcl-2信号通路从而上调pAkt和Bcl-2蛋白有关。     

大麻素anandamide对大鼠海马脑片LTP和海马细胞凋亡的影响

目的 观察大麻素anandamide（AEA）对大鼠海马脑片突触传递长时程增强效应（LTP）及其对海马细胞凋亡的影响.方法 选用Wistar大鼠,迅速断头取脑,剥离海马,制作厚约400μm的脑片,运用电生理的方法观察AEA及其CB1受体拮抗剂AM251对海马脑片CA1区LTP的影响,并采用AO/EB双荧光染色,激光共聚焦显微镜观察AEA和AM251对原代培养大鼠海马神经元凋亡的形态学改变,流式细胞仪技术观察AEA和AM251对原代培养大鼠海马神经元凋亡率的影响.结果 AEA明显抑制大鼠海马脑片LTP的产生,同时AEA有促进原代培养大鼠海马神经元凋亡的作用,而AM251可逆转AEA的上述作用.结论 AEA对大鼠海马脑片CA1区LTP有抑制作用,并且AEA有促使神经元凋亡的神经毒性作用.     

大鼠自体激活雪旺细胞体外诱导骨髓间充质干细胞分化为神经元细胞的观察

目的观察大鼠自体激活雪旺细胞（AASC）体外诱导骨髓间充质干细胞（BMSC）分化为神经元细胞的可行性。方法取出生8周的Wistar大鼠,结扎双侧坐骨神经,进行雪旺细胞（SC）的自体激活,1周后取出坐骨神经,采用组织块法分离培养纯化AASC。冲洗Wistar大鼠股骨髓腔,梯度密度离心贴壁培养法培养BMSC、并鉴定。将AASC与BMSC共培养（共培养组）,以单独培养的BMSC作对照组。培养10d后分别采用Nestin、NF免疫组化染色鉴定BMSC分化的神经元样细胞。结果共培养组培养7d后无论是BMSC的数目还是Nestin、NF表达阳性的BMSC的数目与对照组相比,P均〈0．01。结论大鼠AASC在体外可诱导BMSC向神经元样细胞分化。     

阿魏酸钠对SNP诱导的凋亡海马神经元NF—kB、par-4基因表达的影响

目的探讨阿魏酸钠（sodium ferulate，SF）对一氧化氮（NO）供体硝普钠（SNP）引起的大鼠海马神经元凋亡及NF-KBP65、par-4基因表达的影响。方法采用sD大鼠海马神经元原代培养，经终浓度分别为10、20、40、80、120、160μmol／L的sF预处理后，用50μmol／L的SNP处理24h，采用MTr法检测细胞存活率，Hochest33258荧光染色检测凋亡，Western blot及RT—PCR检测NF．KBP65、par-4基因表达。结果不同剂量sF（10～160μmol／L）预处理6h可显著提高神经元的存活率，减少SNP引起的核固缩、凝聚和碎裂现象；降低NF—KBP65、par-4mRNA及蛋白的表达。结论sF抑制NO供体SNP诱导的海马神经元凋亡，其机制可能与降低促凋亡基因NF—KBP65、par-4表达有关。     

低温停循环后大鼠海马区神经元树突中微管相关蛋白2的变化

目的目前,大血管手术和复杂先心病术中深低温停循环下神经系统的保护仍有较大提升空间。为了探寻神经保护新的思路,我们对不同温度下停循环的大鼠海马组织进行了损伤评估。方法20只雄性SD大鼠随机分为4组:深低温停循环(15~20℃),中低温停循环(20~25℃),浅低温停循环(25~30℃)和假手术组。术后对大鼠海马组织进行了病理评估,对神经元树突中的微管相关蛋白2(microtubule-associated protein 2,MAP2)的表达,进行了实时定量PCR和蛋白免疫印迹分析。血浆中MAP2和S100β的含量也通过ELISA法进行了测定。结果与假手术相比,各组低温停循环大鼠的海马神经元的树突微管和线粒体嵴均有溶解。与假手术组相比,浅低温停循环组海马组织中MAP2的mRNA表达上调,MAP2蛋白组间无差异。各组血浆S100β含量并无统计学差异,而与假手术组相比,浅低温停循环组的血浆MAP2升高。结论低温停循环后,神经元的树突受损,微管溶解,其构成蛋白MAP2释放到血液。促进MAP2生成,减少MAP2的损失可能是一种有效的神经保护策略。     

索曼染毒对大鼠脑组织乙酰胆碱受体m1和m4 mRNA表达的影响

目的研究慢性小剂量索曼(soman)染毒对大鼠海马和前额叶皮层乙酰胆碱受体m1和m4 mRNA表达的影响。方法以皮下注射索曼为大鼠染毒模型,雄性健康Wistar大鼠随机分为6μg/kg染毒组(6μg/kg)、10μg/kg染毒组(10μg/kg)、生理盐水对照组(相同体积的生理盐水),半数致死量对照组(大鼠在取样前注射一次索曼,剂量为100μg/kg),每组6只;除半数致死量染毒对照组外,均每日上午背部注射1次,共14 d。采用RT-PCR方法检测大鼠海马和前额叶皮层乙酰胆碱受体m1和m4 mRNA表达水平。结果乙酰胆碱受体m1和m4 mRNA的变化结果以OD乙酰胆碱受体m1(乙酰胆碱受体m4)/ODGAPDH表示,6μg/kg组大鼠前额叶皮层乙酰胆碱受体m1 mRNA为(0.250±0.016),10μg/kg组为(0.247±0.018),均显著低于生理盐水对照组(0.287±0.021)和半数致死量对照组(0.277±0.028)(P<0.05);海马乙酰胆碱受体m1 mRNA表达无明显变化(P>0.05),6μg/kg组为(0.275±0.022),10μg/kg组为(0.270±0.019),生理盐水对照组为(0.294±0.027),半数致死量对照组为(0.289±0.029)。6μg/kg组和10μg/kg组前额叶皮层乙酰胆碱受体m4 mRNA表达分别为(0.364±0.031)和(0.426±0.066),均显著高于生理盐水对照组(0.274±0.025)和半数致死量对照组(0.271±0.046)(P<0.01),6μg/kg组和10μg/kg组海马乙酰胆碱受体m4 mRNA表达分别为(0.627±0.030)和(0.671±0.074),均显著高于生理盐水对照组(0.528±0.031)和半数致死量对照组(0.531±0.054)(P<0.01)。在生理盐水对照组和半数致死量对照组间乙酰胆碱受体m1 mRNA和m4 mRNA表达水平均无显著差别(P>0.05)。结论小剂量索曼染毒致大鼠前额叶皮层乙酰胆碱受体m1mRNA表达下降,海马和前额叶皮层乙酰胆碱受体m4 mRNA表达增加,这种变化为索曼慢性作用的结果。索曼可能从基因水平影响乙酰胆碱M受体的表达,从而导致胆碱能系统功能下降及大鼠学习记忆障碍。     

衰老和缺锌大鼠海马胆囊收缩素神经元数目及免疫反应强度的…

选用Ｗｉｓｔａｒ雄性大鼠２４只，分为缺Ｚｎ组（７月龄）、老龄组（２８月龄）和对照组（７月龄），每组各８只。采用免疫组化ＡＢＣ结合图象分析，观察海马胆囊收缩素（ＣＣＫ）神经数目及其免疫反应强度的变化。结果表明，与对照组相比，缺Ｚｎ大鼠与老龄大鼠海马ＣＣＫ阳性神经元的数目和免疫反应强度均明显降低（Ｐ〈０．０５或Ｐ〈０．０１）。提示衰老过程中海马ＣＣＫ阳性神经元的数目减少，ＣＣＫ免疫反应强度降低；衰老过     

硝普钠对体外培养的海马神经元NF-κB和caspase-3基因表达的影响

目的探讨NO供体硝普钠（SNP）诱导体外培养的海马神经元凋亡与核因子-kappaB（NF）-κB p65和caspase-3表达变化的关系。方法终浓度分别为0、25、50、100、200、400μmol/L的SNP处理海马神经元24 h,用MTT比色法分析细胞存活率;Hoechst33258荧光染色观察凋亡的形态学改变;DNA琼脂糖凝胶分析凋亡的生化特征;用RT-PCR检测NF-κB p65、caspase-3 mRNA表达变化;Western印迹检测NF-κB p65、caspase-3蛋白表达的变化。结果SNP可剂量依赖性的降低神经元的存活率;荧光显微镜可见染为高亮蓝色的典型凋亡小体,其细胞核明显固缩、凝聚和断裂;电泳图谱显示清晰的DNA梯度。随着SNP剂量的增加,NF-κB p65 mRNA、NF-κB p65蛋白表达逐渐增加;caspase-3 mRNA表达无改变,但caspase-3酶原被裂解活化;从50μmol/LSNP起,caspase-3相对酶活性显著增加,为对照组的3.02倍,100μmol/L达最大值。结论SNP可诱导培养的海马神经元凋亡,其凋亡机制可能与增加NF-κB p65表达及激活caspase-3酶原有关。     

糖尿病脑缺血再灌注大鼠海马CA1区Fas蛋白表达及细胞凋亡的研究

目的观察Fas蛋白在糖尿病脑缺血再灌注大鼠海马CA1区神经元损伤中的表达。方法采用链脲佐菌素(STZ)诱导和线栓法制备糖尿病大脑中动脉闭塞模型(MCAO),应用免疫组化方法和流式细胞术观察糖尿病脑缺血再灌注组与缺血再灌注组海马CA1区Fas表达变化和细胞凋亡情况。结果缺血再灌注组及糖尿病脑缺血再灌注组大鼠海马CA1区Fas阳性染色阳性细胞分别为(21·3±3·1)个/100μm、(51·9±4·2)个/100μm,较正常对照组〔(1·1±1·7)个/100μm〕及假手术组〔(10·3±2·4)个/100μm〕增多(P<0·05);而糖尿病脑缺血再灌注组比缺血再灌注组增加得更明显(P<0·05);糖尿病脑缺血再灌注组海马CA1区Fas蛋白表达上调,细胞凋亡百分数〔(29·34±8·45)%〕明显高于缺血再灌注组〔(17·59±6·38)%〕(P<0·05)。结论糖尿病大鼠脑缺血再灌注损伤后海马细胞发生凋亡,Fas介导的细胞凋亡机制可能是糖尿病加重脑缺血再灌注海马神经元损伤机制之一。     

衰老和缺锌大鼠海马胆囊收缩素神经元数目及免疫反应强度的变化

选用Ｗｉｓｔａｒ雄性大鼠２４只，分为缺Ｚｎ组（７月龄）、老龄组（２８月龄）和对照组（７月龄），每组各８只。采用免疫组化ＡＢＣ法结合图象分析，观察海马胆囊收缩素（ＣＣＫ）神经数目及其免疫反应强度的变化。结果表明，与对照组相比，缺Ｚｎ大鼠与老龄大鼠海马ＣＣＫ阳性神经元的数目和免疫反应强度均明显降低（Ｐ＜０．０５或Ｐ＜０．０１）。提示衰老过程中海马ＣＣＫ阳性神经元的数目减少，ＣＣＫ免疫反应强度降低；衰老过程中机体Ｚｎ水平的降低是触发这种变化的重要原因之一。     

砷对原代培养海马神经细胞的过氧化损伤

目的观察低剂量砷对新生大鼠海马神经细胞过氧化的影响。方法对原代培养的新生大鼠海马神经细胞经提取、纯化和鉴定后分组进行染毒试验。A组（对照组）、B组（砷50μmol/L）、D组（砷100μmol/L）、F组（砷200μmol/L）、H组（砷400μmol/L）,染毒时间为12 h。经超声波粉碎细胞后,测定神经细胞超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）、乙酰胆碱酯酶（TChE）活性和丙二醛（MDA）含量。结果与对照组比较,各染毒组GSH-Px、S0D和TChE活性显著降低（P〈0.05）,CAT活性和MDA含量显著升高（P〈0.05）,具有明显的剂量效应。结论低剂量砷暴露可引起海马神经细胞的过氧化损伤。     

TrkB-mediated activation of geranylgeranyltransferase I promotes dendritic morphogenesis

Dendrite morphogenesis is regulated by neuronal activity or neurotrophins, which may function by activating intrinsic signaling proteins, including Rho family GTPases. Here we report that activity- and brain-derived neurotrophic factor (BDNF)–dependent dendritic morphogenesis requires activation of geranylgeranyltransferase I (GGT), a prenyltransferase that mediates lipid modification of Rho GTPases. Dendritic arborization in cultured hippocampal neurons was promoted by over-expression of GGT, and reduced by inhibition or down-regulation of GGT. Furthermore, GGT was activated by neuronal depolarization or BDNF, both of which promote dendritic arborization, in cultured hippocampal neurons. Moreover, exploration of a novel environment caused activation of GGT in the mice hippocampus, suggesting that neural activity activates GGT in vivo. Interestingly, GGT was physically associated with tropomyosin-related kinase B (TrkB), the receptor for BDNF, and this association was enhanced by depolarization. Disrupting the GGT-TrkB interaction or down-regulating GGT activity attenuated depolarization- or BDNF-induced dendrite development. Finally, the GGT effect on dendrite arborization was prevented by over-expressing Rac1 with the prenylation site deleted or mutated. Thus depolarization- or BDNF-dependent dendrite development may be mediated by GGT-induced prenylation of Rho GTPases.     

CRH-R1 and CRH-R2 differentially modulate dendritic outgrowth of hippocampal neurons

Corticotropin-releasing hormone (CRH) has been implicated to be involved in the development of dendrites in brain. In the present study, we examined the effect of CRH on dendrite outgrowth in primary cultured hippocampal neurons and defined the specific CRH receptor subtype involved. Treatment of neurons with increasing concentration of CRH resulted in an increase in the total dendritic branch length (TDBL) of neurons compared with untreated neurons over 2-4?days period of treatment. These effects can be reversed by the specific CRH-R1 antagonist antalarmin but not by the CRH-R2 antagonist astressin 2B. Treatment of neurons with urocortin II, the exclusive CRH-R2 agonist, significantly decreased TDBL of the cultured neurons. These effects can be reversed by the CRH-R2 antagonist astressin 2B. Our results suggest that CRH-R1 and CRH-R2 differentially modulate the dendritic growth of hippocampal neurons in culture.     

人激肽释放酶重组腺相关病毒对人脐静脉内皮细胞的影响

目的:构建携带人激肽释放酶(kallikrein,KK)基因的重组腺相关病毒载体,并导入体外培养的人脐静脉内皮细胞(HUVEC),观察其对缓激肽(BK)、一氧化氮(NO)、内皮素-1(ET-1)水平和内皮型一氧化氮合酶(eNOS)基因表达的影响。方法:(1)将KK基因定向克隆入腺相关病毒载体质粒pAAV-MCS中,构建成pAAV-KK;(2)分别将pAAV-KK及pAAV-LacZ与另外两种质粒pHelper、pAAV-RC通过脂质体共同转染AAV-293细胞,包装出rAAV-KK和rAAV-LacZ重组腺相关病毒。将rAAV-LacZ原液稀释成不同浓度梯度并感染HT-1080细胞,通过β-半乳糖苷酶染色测定病毒感染滴度,并观察LacZ传代HT-1080细胞的表达情况;(3)将rAAV-KK感染HUVEC,检测其对缓激肽、NO、ET-1水平和eNOS基因表达的影响。结果:(1)成功构建了带有KK目的基因的重组腺相关病毒载体和带有LacZ报告基因的重组腺相关病毒载体(对照载体);(2)rAAV-LacZ重组病毒以感染滴度6.2×107I U/ml感染HT-1080细胞后,LacZ基因能够在连续传代的HT-1080细胞内稳定持续表达;(3)与空白组、LacZ组(对照组)相比,KK感染组的HUVEC细胞培养液中BK[(2.864±1.36)pmol/ml∶(2.782±1.48)pmol/ml∶(6.576±2.08)pmol/ml]、NO[(16.42±1.02)μmol/L∶(16.46±1.25)μmol/L∶(21.28±1.46)μmol/L]水平明显增加,eNOS基因[(0.353±0.031)∶(0.364±0.049)∶(0.423±0.038)]表达明显增强,ET-1[(262.91±17.85)pg/ml∶(263.56±15.87)pg/ml∶(199.48±16.99)pg/ml]水平显著下降(P均0.01)。结论:(1)成功构建了携带有人激肽释放酶基因的重组腺相关病毒,rAAV-KK感染体外培养的人脐静脉内皮细胞;(2)可使细胞分泌BK、NO,以及表达eNOS基因增加,ET-1减少,改善内皮细胞功能。     

早期生长反应因子1特异诱骗寡核苷酸下调富含半胱氨酸蛋白61抑制大鼠血管平滑肌细胞迁移

目的观察早期生长反应因子1特异诱骗寡核苷酸对体外培养的大鼠血管平滑肌细胞中早期生长反应因子1和富含半胱氨酸蛋白61表达的影响,初步探讨早期生长反应因子1特异诱骗寡核苷酸抑制血管平滑肌细胞迁移的机制。方法体外培养血管平滑肌细胞并转染早期生长反应因子1特异诱骗寡核苷酸,采用逆转录聚合酶链反应和免疫组织化学法检测转染后不同时间点对照组与实验组血管平滑肌细胞中早期生长反应因子1及富含半胱氨酸蛋白61的mRNA和蛋白的表达变化,划痕法测定血管平滑肌细胞迁移距离。结果早期生长反应因子1及富含半胱氨酸蛋白61的mRNA和蛋白在对照组、诱骗组及诱骗对照组中均有表达。在不同时间点,诱骗组早期生长反应因子1及富含半胱氨酸蛋白61的mRNA和蛋白的表达均低于对照组(P<0.05)。转染后48 h,对照组、诱骗组及诱骗对照组血管平滑肌细胞的迁移距离分别为105.23±7.81μm、58.65±12.68μm、106.47±7.60μm,诱骗组血管平滑肌细胞的迁移距离明显低于其他两组(P<0.01)。结论对体外培养的血管平滑肌细胞转染早期生长反应因子1特异诱骗寡核苷酸可以抑制早期生长反应因子1及富含半胱氨酸蛋白61的mRNA和蛋白表达,从而抑制血管平滑肌细胞的迁移。     

Ethanol alters BDNF-induced Rho GTPase activation in axonal growth cones

Background: The effects of ethanol on development of postmitotic neurons include altered neurite outgrowth and differentiation, which may contribute to neuropathology associated with fetal alcohol spectrum disorders. We previously reported that ethanol exposure alters axon growth dynamics in dissociated cultures of rat hippocampal pyramidal neurons. Given the important regulatory role of small Rho guanosine triphosphatases (GTPases) in cytoskeletal reorganization associated with axon growth, and reports that ethanol alters whole cell Rho GTPase activity in other cell types, this study explored the hypothesis that ethanol alters Rho GTPase activity specifically in axonal growth cones. Methods: Fetal rat hippocampal pyramidal neurons were maintained in dissociated cultures for 1 day in control medium or medium containing 11 to 43 mM ethanol. Some cultures were also treated with brain‐derived neurotrophic factor (BDNF), an activator of Rac1 and Cdc42 GTPases that promotes axon extension. Levels of active Rho GTPases in growth cones were measured using in situ binding assays for GTP‐bound Rac1, Cdc42, and RhoA. Axon length, growth cone area, and growth cone surface expression of tyrosine kinase B (TrkB), the receptor for BDNF, were assessed by digital morphometry and immunocytochemistry. Results: Although ethanol increased the surface area of growth cones, the levels of active Rho GTPases in axonal growth cones were not affected in the absence of exogenous BDNF. In contrast, ethanol exposure inhibited BDNF‐induced Rac1/Cdc42 activation in a dose‐dependent manner and increased RhoA activation at the highest concentration tested. Similar TrkB expression was observed on the surface of axonal growth cones of control and ethanol‐treated neurons. Conclusions: These results reveal an inhibitory effect of ethanol on growth cone signaling via small Rho GTPases during early stages of hippocampal development in vitro, and suggest a mechanism whereby ethanol may disrupt neurotrophic factor regulation of axon growth and guidance.     

Spatially selective, testosterone-independent remodeling of dendrites in gonadotropin-releasing hormone (GnRH) neurons prepubertally in male rats

Adult GnRH neurons exhibit a stereotypic morphology with a small soma, single axon, and single dendrite arising from the soma with little branching. The adult morphology of GnRH neurons in mice reflects an anatomical consolidation of dendrites over postnatal development. We examined this issue in rat GnRH neurons with biocytin filling in live hypothalamic slices from infant males, as adult littermates and in gonad-intact males, castrated males, and in males with one of three levels of testosterone (T) treatment. Somatic area and total dendritic length were significantly greater in infant males than in adults. Moreover, total numbers of dendrite branches were greater in infant males as compared with adults. The number of higher order branches and the lengths of higher order branches were also greater in infant males than in adults. Most interestingly, in adults a single dendrite arose from the somata, consistently at 180° from the axon. In contrast, prepubertal animals had an average of 2.2 ± 0.2 primary dendrites arising from somata (range, one to seven primary dendrites). Angles relative to the axon at which dendrites in prepubertal males emanated from GnRH somata were highly variable. Castration at 25 d of age and castration at 25 d of age with one of three levels of T treatment did not influence morphological parameters when GnRH neurons were examined between 40 d and 48 d of age. Thus, a spatially selective remodeling of primary dendrites and consolidation of distal GnRH dendritic arbors occurs during postnatal development and is largely independent of T.     

Brain-derived neurotrophic factor mediates estradiol-induced dendritic spine formation in hippocampal neurons

Dendritic spines are of major importance in information processing and memory formation in central neurons. Estradiol has been shown to induce an increase of dendritic spine density on hippocampal neurons in vivo and in vitro. The neurotrophin brain-derived neurotrophic factor (BDNF) recently has been implicated in neuronal maturation, plasticity, and regulation of GABAergic interneurons. We now demonstrate that estradiol down-regulates BDNF in cultured hippocampal neurons to 40% of control values within 24 hr of exposure. This, in turn, decreases inhibition and increases excitatory tone in pyramidal neurons, leading to a 2-fold increase in dendritic spine density. Exogenous BDNF blocks the effects of estradiol on spine formation, and BDNF depletion with a selective antisense oligonucleotide mimics the effects of estradiol. Addition of BDNF antibodies also increases spine density, and diazepam, which facilitates GABAergic neurotransmission, blocks estradiol-induced spine formation. These observations demonstrate a functional link between estradiol, BDNF as a potent regulator of GABAergic interneurons, and activity-dependent formation of dendritic spines in hippocampal neurons.     

钙调蛋白抑制剂对N-甲基-D-天冬氨酸诱导的大鼠皮质神经元损伤的保护作用

目的观察钙调蛋白抑制剂w_7对N-甲基-D-天冬氨酸（NMDA）诱导的皮质神经元损伤的影响,并探讨其可能的机制。方法原代培养sD大鼠（120只）皮质神经元,神经元特异性稀醇化酶（NSE）染色方法确定神经元的比例;添加不同浓度的NMDA,制备细胞损伤模型,MTT法评估细胞生存力;原代大鼠皮质神经元培养后,随机分为对照组、损伤组（NMDA）、不同剂量w-7保护组（25、50、100μmol／L）,MIT法评价细胞生存力;对上述各组进行吖啶橙荧光染色,观察细胞凋亡形态;采用WesternBlot方法,测定上述各组神经元的p38MAPK和NF-kBp65表达情况。结果①以NSE抗体标记培养的神经元,阳性神经元的比例为90．86％。②不同浓度的NMDA对培养大鼠皮质神经细胞具有损伤作用,实验结果表明50μmol／L的NMDA对细胞的损伤以凋亡为主,以此剂量做后续实验。③与对照组比较,损伤组的吸光度（A）值降低,差异有统计学意义,P〈0．01;与损伤组比较,w-7各保护组A值均升高（P〈0．01或P〈0．05）,差异有统计学意义。④吖啶橙荧光染色可观察到凋亡细胞,保护组随着w-7剂量的增加凋亡细胞数量减少。（5）WesternBlot结果显示,w-7可以使p38MAPK和NF-KBp65表达下降,差异有统计学意义（P〈0．05或P〈0．01）。结论w-7可能通过降低p38MAPK和NF．KBp65的途径,对NMDA诱导的神经元损伤起保护作用。